Suggested role of NosZ in preventing N2O inhibition of dissimilatory nitrite reduction to ammonium

ABSTRACT Climate change and nutrient pollution are among the most urgent environmental issues. Enhancing the abundance and/or the activity of beneficial organisms is an attractive strategy to counteract these problems. Dissimilatory nitrate reduction to ammonium (DNRA), which theoretically improves nitrogen retention in soils, has been suggested as a microbial process that may be harnessed, especially since many DNRA-catalyzing organisms have been found to possess nosZ genes and the ability to respire N2O. However, the selective advantage that may favor these nosZ-harboring DNRA-catalyzing organisms is not well understood. Here, the effect of N2O on Nrf-mediated DNRA was examined in a soil isolate, Bacillus sp. DNRA2, possessing both nrfA and nosZ genes. The DNRA metabolism of this bacterium was observed in the presence of C2H2, a NosZ inhibitor, with or without N2O, and the results were compared with C2H2-free controls. Cultures were also exposed to repeated oxic-anoxic transitions in the sustained presence of N2O. The NO2 −-to-NH4 + reduction following oxic-to-anoxic transition was significantly delayed in NosZ-inhibited C2H2-amended cultures, and the inhibition was more pronounced with repeated oxic-anoxic transitions. The possibility of C2H2 involvement was dismissed since the cultures continuously flushed with C2H2/N2 mixed gas after initial oxic incubation did not exhibit a similar delay in DNRA progression as that observed in the culture flushed with N2O-containing gas. The findings suggest a possibility that the oft-observed nosZ presence in DNRA-catalyzing microorganisms secures an early transcription of their DNRA genes by scavenging N2O, thus enhancing their capacity to compete with denitrifiers at oxic-anoxic interfaces. IMPORTANCE Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO3 − and/or NO2 − to NH4 +. Interestingly, DNRA-catalyzing microorganisms possessing nrfA genes are occasionally found harboring nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N2O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N2O may delay the transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA-possessing microorganisms have retained nosZ genes: to remove N2O that may otherwise interfere with the transition from O2 respiration to DNRA.


Supplemental methods and materials
Text S1.Stoichiometric calculation of additional electron-accepting capacity gained by NosZ activity.
Assuming that 95% of NO2 -is reduced to NH4 + via DNRA (Eq. 1) and 5% results in N2O production (Eq.2), the number of moles of electrons transferred to N2O for reduction to N2 would be 0.05 per mole NO2 -consumed, amounting to 0.88% of the number of moles of electrons transferred to NH4 + from 0.95 mole NO2 -(5.7 per mole NO2 -consumed).
NO2 -+ 6e -+ 8H + → NH4 + + 2H2O (1) Text S2.Reverse transcription quantitative PCR (RT-qPCR) method The nrfA and nosZ transcripts in Bacillus sp.DNRA2 cultures were quantified by RT-qPCR using a previously established protocol (1).Immediately after sampling, 1 mL of RNA Protect Bacteria Reagent (Qiagen, Hilden, Germany) was mixed with the 0.5-mL culture in a RNase-free 1.5-mL Safe-Lock tube (Eppendorf, Hamburg, Germany).The cell pellets collected after 5 min centrifugation at 5,000 x g were stored at -80 °C until use.The pellets were thawed on ice, and 350 µL buffer RLT was added to the thawed tube along with 40 mg of 0.1 mm diameter glass beads (Omni International, Kennesaw, GA).The cells were disrupted using an Omni Bead Ruptor 12 homogenizer (Kennesaw, GA), and total RNA was extracted using RNeasy Mini Kit (Qiagen) following the protocol provided by the manufacturer.After digestion with RNase-Free DNase Set (Qiagen), RNA was purified with RNeasy MinElute Cleanup kit (Qiagen).The absence of genomic DNA in the eluent was later confirmed by performing PCR targeting the recA gene with a fraction of the eluent stored at -80 °C.Reverse transcription of total RNA was performed using Superscript® III Reverse Transcriptase (Invitrogen, Carlsbad, CA).The remaining RNA was removed using RNase H (Invitrogen), and the resulting cDNA solution was diluted 5-fold with nuclease-free water (Invitrogen) and stored at -20 °C.The qPCR assays were performed using SYBR Green detection chemistry, using the primer sets designed de novo from the nrfA and nosZ gene sequences downloaded from the NCBI's genome database (Accession number: NZ_JABAIT000000000) with Primer3 software (Table S3; Rozen and Skaletsky, 2000).The single-copy housekeeping gene recA was also quantified, as this gene is known to be constitutively expressed at a relatively constant level, and thus has been used for normalization of RT-qPCR data in previous studies (2,3).The information regarding the primer sets is summarized in Table S3.
Quantitative PCR was performed on a QuanStudio 3 real-time PCR instrument (Thermo Fisher Scientific, Waltham, MA).For each target gene, a calibration curve was constructed with a 108-101 copies µL-1 dilution series of the amplicons inserted in PCR®2.1 vectors.

Table S1 .
Inventories of nrfA/nosZ genes in sequenced Bacillus genomes The values in the parentheses are the standard deviations of the measurements from biological triplicates 71 a a